Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component in the specific recognition of target cells by NK cells are NK cell inhibitory receptors that recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents the killing of normal, healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors block NK cell activation. Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses. NK cell cytotoxicity is achieved by polarized release of perforin-containing granules towards target cells. As polarization and degranulation can be controlled by separate signals, their respective sensitivity to inhibitory receptors and the requirements for inhibition were evaluated. Expression of HLA-C or HLA-E on the human cell line 221 blocked granule polarization, degranulation, and CD16-dependent MIP-1alpha secretion by NK cell clones with inhibitory receptors of matching HLA specificity. However, HLA-C or HLA-E on Drosophila S2 cells did not fully inhibit CD16-dependent degranulation and MIP-1alpha secretion, suggesting that other receptor-ligand interactions, which occur during contact with 221 cells, are required for complete inhibition. In contrast, HLA-C or HLA-E on S2 cells were sufficient to block granule polarization induced by LFA-1 or by NKG2D. Therefore, engagement of inhibitory receptors by HLA class I on target cells is sufficient to block different signals for granule polarization, but not degranulation. Many cellular responses, such as autoimmunity and cytotoxicity, are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIM). We have shown that binding of inhibitory NK cell receptors to HLA class I on target cells induced tyrosine phosphorylation of the adapter Crk, concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore, Crk dissociated from the guanine exchange factor C3G and bound to tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity, providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex, observed here with two types of inhibitory receptors, expands the signaling potential of the large ITIM-receptor family, and reveals an unsuspected component of the inhibitory mechanism. Interleukin (IL)-15 is presented in trans, bound to the alpha chain of the IL-15 receptor (IL-15Ralpha) on presenting cells, to cells bearing the receptor beta and gamma chains. The confinement of IL-15 stimulation to sites of cell-to-cell contact has potential for regulation by other receptors. We have used primary human NK cells to test the sensitivity of IL-15 transpresentation to NK cell inhibitory receptors. Human target cells expressing ligands for different inhibitory receptor were transfected with IL-15Ralpha. Proliferation of NK cells and phosphorylation of ribosomal protein S6 in response to transpresented IL-15 were reduced by coengagement of inhibitory receptors. Therefore, transpresentation of IL-15 is subject to regulation by MHC class I-specific inhibitory receptors. These conclusions were reached by studying the response of primary, freshly isolated, human NK cells to human cells transfected with IL-15Ralpha, reveal an unsuspected level of regulation in the response of NK cells to the cytokine IL-15, which is essential for their development, survival, and proliferation. These findings demonstrate a novel mechanism to attenuate NK cell responses to IL-15 transpresentation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.